Characterizing Noncovalent Therapeutics in Biomatrices by Native Affinity Capture Mass Spectrometry:...

Protein therapeutic complexes, such as interchain cysteine-conjugated antibody-drug conjugates (ADCs) and siRNA-based antibody-oligonucleotide conjugates (siRNA AOCs) that form noncovalent antibody subunit assemblies, are increasingly prevalent. Biotransformation assessments of protein therapeutics in complex biomatrices are often conducted using affinity capture coupled with reversed-phase liquid chromatography-mass spectrometry under denaturing conditions (AC-MS, for clear distinction referred to as AC-denaturing MS), which allows direct measurement of clipping sites and relative quantification. However, AC-denaturing MS is unsuitable for analyzing the biotransformation of noncovalent protein therapeutics, as stability information is lost for the noncovalent component. This manuscript presents a novel biotransformation analysis method that preserves noncovalent interactions by combining affinity capture with neutral pH competitive elution and native size-exclusion chromatography-mass spectrometry, named native affinity capture-mass spectrometry (native AC-MS). The successful recovery of noncovalent complexes, including a cysteine-conjugated ADC mimic molecule and siRNA AOCs in various biomatrices, demonstrates the capability of native AC-MS to maintain noncovalent interactions during characterization. Optimized competitive elution exhibits a recovery comparable to that of standard denaturing elution. Native AC-MS offers sufficient sensitivity (low μg/mL limit of detection for the antibody) for in vivo studies, as demonstrated by an in vivo plasma siRNA AOC analysis example. To our knowledge, this is one of the first reports of the in vivo biotransformation analysis of siRNA AOCs. The method provides valuable information, including relative abundance and metabolite identification, and can be widely adapted for biotransformation of noncovalent modalities.